Fluorescent compounds can be used to directly stain or label a sample so that the sample can be identified and quantitated. Fluorescent dyes are widely used in biological applications in which a highly sensitive detection reagent is desirable. For example, fluorescent dyes may be added as part of an assay for a biological target analyte. Dyes that are able to preferentially bind to a specific biological component in a sample can be used to determine the presence or quantity of that specific ingredient.
Fluorescent dyes with longer wavelength absorption and emission are particularly useful in conjunction with materials of biological origin such as cells and tissues, where background or inherent fluorescence or absorption often interferes with detection of the added fluorescent dye. Furthermore, biological specimens often have decreasing levels of both absorption and fluorescence emission as the illumination energy approaches the infrared.
Certain fluorescent compounds have a strong affinity for DNA and can serve as a fluorescent marker for DNA. Fluorescent markers for DNA find use in various applications, including fluorescence microscopy and flow cytometry. A commonly used fluorescent compound that is known to have an affinity for double-stranded DNA is 7-aminoactinomycin D (7-AAD). 7-AAD can be efficiently excited using standard helium-neon and argon lasers, laser diodes or arc lamps or other such focused light sources (absorption maximum at 546 nm) and exhibits a large Stokes shift (emission maximum at 647 nm), which makes it compatible with many types of fluorophores and useful in multicolor applications. 7-AAD is widely used as a nuclear stain for dead cells, since it does not readily pass through intact cell membranes. In cell viability assays, cells with compromised membranes will stain with 7-AAD, while live cells with intact cell membranes will not. A limitation of using 7-AAD as a nuclear stain is that the staining process (i.e., the time required for 7-AAD to associate with the DNA) requires a relatively long incubation time. For example, for dead cell identification, the staining process typically takes about 30 minutes to fully equilibrate. Long incubation times hamper the use of 7-AAD in high throughput applications and other applications requiring rapid staining or analysis. Additionally, the DNA content cell cycle histogram generated using fixed cells stained with 7-AAD exhibits a high degree of variability, as measured by the % Coefficient of Variation (% CV) of the G0G1 peak of the histogram.
Thus, there exists a need for fluorescent compounds that selectively and rapidly stain cells with compromised cell membranes and have excitation spectra in the visible region and emission spectra in the near infrared (IR) region of the electromagnetic spectrum.